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Application of phospholipid scramblase 1 in preparing anti-hepatitis B virus infective medicament

A technology of phospholipid crawling enzymes and uses, which is applied in antiviral agents, pharmaceutical formulas, medical preparations containing active ingredients, etc., to achieve important social and economic benefits

Inactive Publication Date: 2010-08-04
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no literature report about the anti-hepatitis B virus effect of phospholipid crawlase 1

Method used

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  • Application of phospholipid scramblase 1 in preparing anti-hepatitis B virus infective medicament
  • Application of phospholipid scramblase 1 in preparing anti-hepatitis B virus infective medicament
  • Application of phospholipid scramblase 1 in preparing anti-hepatitis B virus infective medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Inhibition of Intracellular Expression of PLSCR1 on HBV Replication

[0035] Materials and Methods

[0036] 1. Cell culture

[0037] The cells used are the liver cancer cell line HepG2 cell line and the HepG2.2.15 cell line transfected with HBV DNA. HepG2.2.15 cells are derived from HepG2 cells, contain integrated HBV DNA, and can continuously and stably secrete Dane's granules, HBsAg, HBV DNA, etc. into the culture medium during cell culture. HepG2 cells were cultured with DMEM cell culture medium containing 10% fetal bovine serum (FBS, Gibco), and HepG2.2.15 cells were cultured with MEM cell culture medium containing 10% fetal bovine serum, 380 μg / ml G418 (Promega).

[0038] 2. Plasmid

[0039] The pCMV-HA-PLSCR1 plasmid was constructed by our laboratory, and primers were designed according to the PLSCR1 sequence (NM_021105.2) published by NCBI to amplify the PLSCR1 CDS region, which was inserted into the pCMV-HA plasmid by cloning, screened by positive c...

Embodiment 2

[0059] Example 2: Inhibition of HBV replication by adding exogenous recombinant GST-PLSCR1 protein

[0060] Materials and Methods

[0061] 1. Construction of pGEX-4T-1-PLSCR1 plasmid

[0062] pGEX-4T-1-PLSCR1 was constructed in our laboratory, and primers were designed according to the PLSCR1 sequence (NM_021105.2) published by NCBI to amplify the PLSCR1 CDS region, which was inserted into the pGEX-4T-1 plasmid by cloning, and positively cloned Screening, sequencing, and BLAST comparison results showed that the constructed pGEX-4T-1-PLSCR1 was completely correct.

[0063] 2. Induced expression of GST-PLSCR1 fusion protein

[0064] Inoculate Escherichia coli DH5α transformed with the vector pGEX-4T-1-PLSCR1 expressing GST fusion protein into LB medium, shake overnight at 37°C, then inoculate into fresh LB medium according to 2% inoculum size, and culture at 30°C After 5 hours, IPTG was added to a final concentration of 0.1 mmol / L-1 mmol / L, and induction culture was continued...

Embodiment 3

[0090] Example 3: Inhibition of HBV replication by adding exogenous recombinant His-PLSCR1 fusion protein

[0091] Materials and Methods

[0092] 1. Construction of Pet28a-PLSCR1 plasmid

[0093] Pet28a-PLSCR1 was constructed in our laboratory. Primers were designed according to the PLSCR1 sequence (NM_021105.2) published by NCBI to amplify the CDS region of PLSCR1, which was inserted into the Pet28a plasmid by cloning. The positive clones were screened, sequenced, and compared by BLAST The results showed that the constructed Pet28a-PLSCR1 was completely correct.

[0094] 2. Induced expression and purification of His-PLSCR1 fusion protein

[0095] Inoculate the Escherichia coli containing the recombinant expression plasmid on an agar plate, pick a single colony from a fresh agar plate, inoculate 2ml LB medium, add appropriate antibiotics (final concentration 100mg / L) in a 20ml culture tube, shake Cultivate overnight on the bed, dilute the bacteria overnight at a ratio of 1:...

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Abstract

The invention relates to novel application of a biochemical substance in pharmaceutical engineering, in particular to application of phospholipid scramblase 1 (PLSCR1) in preparing a medicament for treating and preventing hepatitis B virus (HBV) infection related diseases. The laboratory discovers that the PreS1 structure domain of the PLSCR1 and the HBV surface protein has the characteristic of association by screening of yeast two-hybrid technology. Further research proves that the protein can effectively prohibit replication and expression of the hepatitis B virus in cultured HepG2.2.15 cells and HBV1.3 transfected HepG2 cell models. Therefore, the invention relates to the PLSCR1 protein which becomes the novel medicament for treating hepatitis B virus related diseases to reduce harmfulness of the hepatitis B virus related diseases.

Description

technical field [0001] The present invention relates to a new application of biochemical substances in pharmaceutical engineering, more specifically, phospholipid scramblase 1 (PLSCR1) is used in the preparation of medicines for treating and preventing hepatitis B virus (HBV) infection-related diseases in the application. Background technique [0002] Hepatitis B virus (HBV) infection is a serious threat to human health and is an important cause of hepatitis, liver cirrhosis, and liver cancer. There are 350 million hepatitis B virus carriers in the world, and about 120 million HBV carriers in China, of which more than 20 million are chronic hepatitis B patients. The direct treatment cost of chronic viral hepatitis in my country exceeds 50 billion RMB every year. HBV infection not only causes huge economic and spiritual burdens to patients and their families, but also causes huge labor and economic losses to the country, and even leads to serious social problems such as emp...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P31/20
Inventor 王升启杨静朱向前苏婧丁晓然王学军胡伟
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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